Introduction: Cell therapy has been widely considered as a therapeutic approach for neurodegenerative diseases and nervous system damage. Cholinergic neurons as one of the most important neurons that play a significant role in controlling emotions, mobility, and autonomic systems. In this study, human dental pulp stem cells (hDPSCs) were differentiated into the cholinergic neurons by β-mercaptoethanol in the preinduction phase and also by the nerve growth factor (NGF) in the induction phase. 
Methods: The hDPSCs were evaluated for CD73, CD31, CD34, and Oct-4. Concentration-time relationships for NGF were assessed by evaluating the viability rate of cells and the immune response to nestin, neurofilament 160, microtubule-associated protein-2, and choline acetyltransferase.
Results: The hDPSCs had a negative response to CD34 and CD31. The optimal dose for the NGF was 50 ng/mL seven days after the induction when the highest percentage of expressing markers for the cholinergic neurons (ChAT) was detected.
Conclusion: The results of this study provided a method for producing cholinergic neurons by hDPSCs, which can be used in cytotherapy for degenerative diseases of the nervous system and also spinal cord injury.